A number of CRD enzymes are known (see Yuki Gosei Kagaku, 49, 52 (1991) and Eur. J. Biochem., 184, 1 (1981)). Among such CRD enzymes, those which act on 4-halo acetoacetic ester to produce (S)-4-halo-3-hydroxy butyric ester, which are derived from microbes, and which have reported characteristics, are only a Geotrichum candidum derived enzyme (Enzyme Microb. Technol. (1992), Vol. 14, 731) and a Candida parapsilosis derived enzyme (Enzyme Microb. Technol. (1993), Vol. 15, 950). However, no information has been reported on genes coding these two types of enzymes. The reduction of 4-halo acetoacetic ester using such enzymes only proceeds at a low substrate concentration. It is therefore impractical to synthesize (S)-4-halo-3-hydroxy butyric ester using such enzymes as catalysts.
Besides the above reaction using the two types of enzymes, a number of reactions using microbe bodies and the products of such reactions are known to realize asymmetric reduction of 4-halo acetoacetic ester (Japanese Patent No. 1723728, Japanese Laid-Open Publication Nos. 6-209782 and 6-38776, etc.) However, such reactions are not performed at a high substrate concentration, and thus it cannot be asserted that a practical production method has been established. See, for example, a reaction method using a two-phase system with an organic solvent (Japanese Patent No. 2566962). A method using a ruthenium-optically active phosphine complex as a catalyst has also been reported (Japanese Laid-Open Publication No. 1-211551). This method however has many problems to be solved, such as the requirement of a high-pressure reaction vessel and need for an expensive catalyst.
Under the above circumstances, development of a practical enzyme has been desired for use in asymmetric reduction of a carbonyl compound such as 4-halo acetoacetic ester to produce an optically active alcohol such as (S)-4-halo-3-hydroxy butyric ester.
A CRD enzyme requires a reduction-type coenzyme for reaction. Conventionally, when a carbonyl compound is to be reduced using a microbe body and the like having a CRD enzyme, a saccharide such as glucose is added to a reaction system to activate a group of regeneration-system enzymes for changing an oxidized coenzyme to a reduced type, thereby regenerating the coenzyme so as to be used for the reduction. Such a group of regeneration-system enzymes are likely to be blocked or damaged by substrates and reduced products. This has been considered to be one of major reasons why the reduction proceeds only when the concentration of substrates or products is low. It is known that the amount of an expensive coenzyme used during reduction can be greatly reduced by combining an enzyme having the ability of regenerating a coenzyme on which a CRD enzyme depends with the CRD enzyme during the reaction (Japanese Patent No. 2566960 and Enzyme Microb. Technol. (1993), Vol. 15, 950, for example). In this case, however, it is required to prepare an enzyme source for regenerating the coenzyme separately from the preparation of the CRD enzyme before the regenerating enzyme is added to a reaction system.
The Inventors of the present application have discovered a novel Candida-genus derived CRD enzyme, and found that an optically active alcohol can be efficiently produced from a carbonyl compound by using this CRD enzyme.
Also found is that an optically active alcohol can be efficiently produced by using a transformed cell containing a gene of an enzyme having the ability of regenerating acoenzyme (e.g., a glucose dehydrogenase gene) concurrently.
Thus, the present invention to be described in the specification can advantageously provide a novel CRD enzyme, a DNA coding this enzyme, a plasmid having this DNA, a transformant which is a cell transformed with this plasmid, and a production method of an optically active alcohol using the above enzyme and/or transformed cell.